Timothy D. O'Connell

Isolation and Culture of Adult Mouse Cardiac Myocytes

Cardiac myocytes are activated by hormonal and mechanical signals and respond in a variety of ways, from altering contractile function to inducing cardio-protection and growth responses. The use of genetic mouse models allows one to examine the role of cardiac-specific and other genes in cardiac function, hypertrophy, cardio-protection, and diseases such as ischemia and heart failure. However, studies at the cellular level have been hampered by a lack of suitable techniques for isolating and culturing calcium-tolerant, adult mouse cardiac myocytes. We have developed a straightforward, reproducible protocol for isolating and culturing large numbers of adult mouse cardiac myocytes. This protocol is based on the traditional approach of retrograde perfusion of collagenase through the coronary arteries to digest the extracellular matrix of the heart and release rod-shaped myocytes. However, we have made modifications that are essential for isolating calcium-tolerant, rod-shaped adult mouse cardiac myocytes and maintaining them in culture. This protocol yields freshly isolated adult mouse myocytes that are suitable for biochemical assays and for measuring contractile function and calcium transients, and cultured myocytes that are suitable for most biochemical and signaling assays, as well as gene transduction using adenovirus.

Overview of the Alliance for Cellular Signaling

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

An α1A-Adrenergic–Extracellular Signal-Regulated Kinase Survival Signaling Pathway in Cardiac Myocytes

Background— In α1-AR knockout (α1ABKO) mice that lacked cardiac myocyte α1-adrenergic receptor (α1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte α1-ARs and whether the α1A, α1B, or both subtypes mediated protection. Therefore, we investigated α1A and α1B subtype–specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of α1-ARs in cardiac myocytes. Methods and Results— We cultured α1ABKO myocytes and reconstituted α1-AR signaling with adenoviruses expressing α1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H2O2 and was measured by annexin V/propidium iodide staining. In α1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the α1A subtype, but not the α1B, rescued α1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined α1-AR activation of extracellular signal-regulated kinase (ERK). In α1ABKO hearts, aortic constriction failed to activate ERK, and in α1ABKO myocytes, expression of a constitutively active MEK1 rescued α1ABKO myocytes from norepinephrine-induced death. In addition, only the α1A-AR activated ERK in α1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked α1A survival signaling in α1ABKO myocytes. Conclusions— Our results demonstrate a direct protective effect of the α1A subtype in cardiac myocytes and define an α1A-ERK signaling pathway that is required for myocyte survival. Absence of the α1A-ERK pathway can explain the failure to activate ERK after aortic constriction in α1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.

Omega-3 Fatty Acids Prevent Pressure Overload–Induced Cardiac Fibrosis Through Activation of Cyclic GMP/Protein Kinase G Signaling in Cardiac Fibroblasts

Background— Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis. Methods and Results— We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction–induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-&bgr;1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-&bgr;1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. Conclusion— Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-&bgr;1–induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.

Navigating the signalling network in mouse cardiac myocytes

Cardiac myocytes have a complex network of signals that regulates their essential role in the rhythmic pumping of the heart. This network is an appealing model system in which to study the basic principles underlying cellular signalling mechanisms. Progress in this effort has come through the establishment of standardized myocyte isolation and culture procedures and characterization of important signalling responses.

Nuclear α1-Adrenergic Receptors Signal Activated ERK Localization to Caveolae in Adult Cardiac Myocytes

We previously identified an α1-AR-ERK (α1A-adrenergic receptor–extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of α1-AR subtypes (α1A and α1B) and how their localization influences α1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent α1-AR antagonist, we localized endogenous α1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify α1 subtype localization, we reconstituted α1 signaling in cultured α1A- and α1B-AR double knockout cardiac myocytes using α1-AR–green fluorescent protein (GFP) fusion proteins. Similar to endogenous α1-ARs and α1A- and α1B-GFP colocalized with LAP2 at the nuclear membrane. α1-AR nuclear localization was confirmed in vivo using α1-AR-GFP transgenic mice. The α1-signaling partners Gαq and phospholipase Cβ1 also colocalized with α1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that α1-mediated activation of ERK was not inhibited by a membrane impermeant α1-blocker, suggesting α1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe α1-AR localization to caveolae, but we found that α1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear α1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.

Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure.

IMMUNOCHEMICAL IDENTIFICATION OF THE 1,25-DIHYDROXYVITAMIN D3 RECEPTOR PROTEIN IN HUMAN HEART

A number of recent clinical observations suggest that vitamin D3plays an important role in maintaining normal cardiovascular function, either directly through its receptor in cardiac muscle, or indirectly through its infuence on circulating levels of calcium or on other regulatory factors. By using an antibody directed against the recombinant vitamin D3receptor, we have identified the receptor protein for 1,25(OH)2D3in tissue from two human hearts. The identification of the 1,25(OH)2D3receptor in human heart lends credence to the hypothesis that 1,25(OH)2D3directly effects the human heart and may be involved in several clinically relevant pathological conditions involving the vitamin D3endocrine system.

Diminished GATA4 Protein Levels Contribute to Hyperglycemia-induced Cardiomyocyte Injury

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. The transcription factor GATA4 is essential for cardiac homeostasis, and its protein levels are dramatically reduced in the heart in response to diverse pathologic stresses. In this study, we investigated if hyperglycemia affects GATA4 expression in cardiomyocytes and if enhancing GATA4 signaling could attenuate hyperglycemia-induced cardiomyocyte injury. In cultured rat cardiomyocytes, high glucose (HG, 25 or 40 mm) markedly reduced GATA4 protein levels as compared with normal glucose (NG, 5.5 mm). Equal amount of mannitol did not affect GATA4 protein expression (NG, 100 ± 12%; mannitol, 97 ± 8%, versus HG, 43 ± 16%, p < 0.05). The GATA4 mRNA content, either steady-state or polysome-associated, remained unchanged. HG-induced GATA4 reduction was reversed by MG262, a specific proteasome inhibitor. HG did not activate the ubiquitin proteasome system (UPS) in cardiomyocytes as indicated by a UPS reporter, nor did it increase the peptidase activities or protein expression of the proteasomal subunits. However, the mRNA levels of ubiquitin-protein isopeptide ligase (E3) carboxyl terminus of Hsp70-interacting protein (CHIP) were markedly increased in HG-treated cardiomyocytes. CHIP overexpression promoted GATA4 protein degradation, whereas small interfering RNA-mediated CHIP knockdown prevented HG-induced GATA4 depletion. Moreover, overexpression of GATA4 blocked HG-induced cardiomyocyte death. Also, GATA4 protein levels were diminished in the hearts of streptozotocin and db/db diabetic mice (44 ± 7% and 67 ± 13% of control, p < 0.05), which correlated with increased CHIP mRNA abundance. In summary, increased GATA4 protein degradation may be an important mechanism that contributes to hyperglycemic cardiotoxicity.

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.